Flow Cytometric Assessment of Erythrocyte Shape through Analysis of FSC Histograms: Use of Kurtosis and Implications for Longitudinal Evaluation

Sphericity of erythrocytes can be estimated from analysis of FSC signal distribution in flow cytometry. Previously, Pearson’s coefficient of dissymmetry (PCD) and spherical index (SphI) were applied to determine erythrocyte sphericity from the FSC histogram. The aim of the present study is to illustrate the application of kurtosis as an indicator of erythrocyte sphericity in flow cytometry in a broad range of FSC distributions. Moreover, the possibility of longitudinal evaluation of erythrocyte sphericity is studied. Change of erythrocyte sphericity of 10 healthy subjects was induced by variation of buffer osmolarity to validate applicability of sphericity measures. Agreement between the sphericity indicators was then studied in samples from 20 healthy donors taken at three time points, which were processed through density gradient centrifugation and incubated with FITC-labelled antibodies to induce a broad variation of erythrocyte form (1086 samples). SphI, PCD and kurtosis of FSC distribution were calculated. Correlation of the respective measures, standard error of measurement (SEM) and r ratio (intra- to interindividual variance) were determined to illustrate agreement between the sphericity indicators. In the first study part, all sphericity indicators illustrated change of erythrocyte shape as induced by osmolarity variation. In the second part, correlation between kurtosis and SphI was −0.97 and correlation between kurtosis and PCD was 0.58 (p<0.05). In isotype control samples, correlation between kurtosis and SphI was −0.98 and correlation between kurtosis and PCD was 0.48 (p<0.05). In these samples, mean kurtosis was −0.80 (SEM 0.03), mean SphI was 2.19 (SEM 0.04) and mean PCD was −0.31 (SEM 0.02). r ratios of all measures of sphericity were <0.6. Our results show that kurtosis is closely correlated with SphI in a broad range of erythrocyte FSC distributions. Moreover, all measures of sphericity feature r ratios <0.6, highlighting that erythrocyte sphericity appears as a feasible parameter for individual longitudinal data monitoring.